β-Galactofuranoside glycoconjugates on conidia and conidiophores of Aspergillus niger

Galactose in the furanoic conformation appears to be limited to bacteria and lower eukaryotes. Galactofuranoic (Galf)-containing glycoconjugates that occur in organisms pathogenic or allergenic to man are frequently antigenic and immunodominant. We have used an immunochemical approach, employing a monoclonal antibody that recognises Galf epitopes, to investigate the presence of Galf-containing glycoconjugates within conidia and conidiophores of Aspergillus niger. ELISA and immunofluorescence microscopy indicated that specific and saturable binding sites were found on both. Inhibition studies confirmed that this binding was to Galf-containing glycoconjugates. Interestingly, the conidiophore heads were particularly rich in these glycoconjugates. Western blotting identified a Galf glycoprotein of 150-200 kDa from disrupted conidia.     

An unambiguous microassay of galactofuranose residues in glycoconjugates using mild methanolysis and high pH anion-exchange chromatography

An original, unambiguous microassay of galactofuranose (Galf) residues in glycoconjugates is described. The method involves mild acid methanolysis (5 mM HCl) for 3 h at 84 degrees C followed by high pH anion-exchange chromatography using a routine monosaccharide system. The methanolysis products Mealpha-Galf and Mebeta-Galf were characterized chromatographically by comparison with the authentic compounds and by their response to treatment with mild acid and with beta-galactofuranosidase. Testing against p-nitrophenyl-beta-Galf and UDPalpha-Galf showed the method to be applicable to both alpha- and beta- galactofuranosides over the range 10-200 pmol. The results of partial mild methanolysis over shorter periods were consistent with initial inversion of anomeric configuration at methylation followed by anomerization to an equilibrium mixture of alpha- and beta-forms. When applied to a sample of invertase from Aspergillus nidulans, the method indicated that all of the mild acid-labile galactose (78% of the total galactose present) was in the form of a galactofuranoside and that much of this was in the beta-configuration. As expected, when applied to asialofetuin (known to contain galactose only in the pyranoside form, Galp), NPalpha-Galp, NPbeta-Galp, or UDPalpha-Galp, mild acid methanolysis failed to produce any galactofuranoside.     

Fungal α-arabinofuranosidases of glycosyl hydrolase families 51 and 54 show a dual arabinofuranosyl- and galactofuranosyl-hydrolyzing activity

Abstract Aspergillus niger possesses a galactofuranosidase activity, however, the corresponding enzyme or gene encoding this enzyme has never been identified. As evidence is mounting that enzymes exist with affinity for both arabinofuranose and galactofuranose, we investigated the possibility that α-l-arabinofuranosidases, encoded by the abfA and abfB genes, are responsible for the galactofuranosidase activity of A. niger. Characterization of the recombinant AbfA and AbfB proteins revealed that both enzymes do not only hydrolyze p-nitrophenyl-α-l-arabinofuranoside (pNp-α-Araf) but are also capable of hydrolyzing p-nitrophenyl-β-d-galactofuranoside (pNp-β-Galf). Molecular modeling of the AbfB protein with pNp-β-Galf confirmed the possibility for AbfB to interact with this substrate, similarly as with pNp-α-Araf. We also show that galactomannan, a cell wall compound of A. niger, containing β-linked terminal and internal galactofuranosyl moieties, can be degraded by an enzyme activity that is present in the supernatant of inulin-grown A. niger. Interestingly, purified AbfA and AbfB did not show this hydrolyzing activity toward A. nigergalactomannan. In summary, our studies demonstrate that AbfA and AbfB, α-l-arabinofuranosidases from different families, both contain a galactofuranose (Galf)-hydrolyzing activity. In addition, our data support the presence of a Galf-hydrolase activity expressed by A. niger that is capable of degrading fungal galactomannan.     

An extracellular β-galactofuranosidase from Aspergillus niger and its use as a tool for glycoconjugate analysis

Aspergillus niger produces an extracellular β-galactofuranosidase, which can specifically hydrolyse β-D-galactofuranose (Galf) from glycoconjugates. The production of this enzyme can be induced by the addition of a Galf-containing A. niger mycelial wall extract. However, on other carbon sources accumulation occurred only during the starvation conditions of the late stationary phase. Extracellular glucoamylases from this stage of cultivation possessed significantly lower levels of Galf than those from the earlier exponential growth phase when β-galactofuranosidase is absent, suggesting in situ β-galactofuranosidic hydrolysis. The β-galactofuranosidase responsible was subsequently purified to homogeneity and characterised. It is a glycoprotein of 90 kDa (determined by SDS–PAGE) with activity against β-linked Galf residues, with a K_m of 4 mM against p-nitrophenyl-β-D-galactofuranoside and a pH optimum of 3–4. The preparation did not contain other contaminating glycosidase activities; p-nitrophenyl-β-D- and -α-D-galactopyranose, and α-D-methyl-Galf were not hydrolysed. Results are presented to show that this enzyme could be employed as a useful tool for the analysis of glycoconjugates containing biologically important Galf components.     

A GM2-specific beta-hexosaminidase from the roe of striped mullet (Mugil cephalus)

The roe of striped mullet (Mugil cephalus) was found to contain a beta-hexosaminidase different from the beta-hexosaminidases isolated from other sources. The enzyme from mullet roe is able to cleave GalNAc from GM2 without the assistance of either an activator protein or a detergent. It also cleaves the oligosaccharide derived from GM2 and other oligosaccharides containing the GM2 sequence GalNAc beta 4(NeuAc alpha 3)Gal-. However, it is not effective in hydrolyzing neutral glycosphingolipids containing terminal GalNAc or GlcNAc, such as GbOse4Cer, GgOse3Cer, or LcOse3Cer. These results indicate that mullet roe beta-hexosaminidase can specifically cleave GalNAc from the glycoconjugates containing the GM2 sequence. No beta-hexosaminidase with such specificity has been previously described. Thus, this unique enzyme should be very useful for the detection and analysis of glycoconjugates containing the oligosaccharide chains with GM2 sequence.     

Galactofuranose in eukaryotes: aspects of biosynthesis and functional impact

Galactofuranose (Galf) is the five-membered ring form of galactose. It is widely distributed among several branches of the eukaryotic kingdom. This review highlights recent advances in our understanding of the biosynthesis and function of Galf-containing glycoconjugates in fungal Aspergillus spp. and the protozoan trypanosomatid parasites. We give an overview of the biosynthetic pathways leading to the production of glycolipids, glycoproteins and polysaccharides containing Galf in these species and their biological relevance. Remarkably, modification of the cell surface caused by Galf absence often results in morphological abnormalities and an impaired cell wall function in these organisms. Galf-deficient mutants are generally hypersensitive to drugs, exhibit a constitutive osmotic stress phenotype and/or have an attenuated virulence. Since Galf has never been found in mammals and higher plants, Galf-biosynthetic pathways have raised much interest as targets for drug development to combat microbial infections.     

Chemical mechanism of UDP-galactopyranose mutase from Trypanosoma cruzi: a potential drug target against Chagas' disease

UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose (Galf). Galf is found in several pathogenic organisms, including the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Galf) is important for virulence and is not present in humans, making its biosynthetic pathway an attractive target for the development of new drugs against T. cruzi. Although UGMs catalyze a non-redox reaction, the flavin must be in the reduced state for activity and the exact role of the flavin in this reaction is controversial. The kinetic and chemical mechanism of TcUGM was probed using steady state kinetics, trapping of reaction intermediates, rapid reaction kinetics, and fluorescence anisotropy. It was shown for the first time that NADPH is an effective redox partner of TcUGM. The substrate, UDP-galactopyranose, protects the enzyme from reacting with molecular oxygen allowing TcUGM to turnover ～1000 times for every NADPH oxidized. Spectral changes consistent with a flavin iminium ion, without the formation of a flavin semiquinone, were observed under rapid reaction conditions. These data support the proposal of the flavin acting as a nucleophile. In support of this role, a flavin-galactose adduct was isolated and characterized. A detailed kinetic and chemical mechanism for the unique non-redox reaction of UGM is presented.     

Detection of beta-galactofuranosidase production by Penicillium and Aspergillus species using 4-nitrophenyl beta-D-galactofuranoside

An assay was developed for detecting beta-galactofuranosidase produced by Penicillium and Aspergillus spp. The substrate for the assay, 4-nitrophenyl beta-D-galactofuranoside, was synthesized from penta-O-acetyl-beta-D-galactofuranose and 4-nitrophenol by a tin chloride catalyzed reaction followed by O-deacetylation. Aspergillus spp. produced only small quantities of beta-galactofuranosidase during 30 d at 25 degrees C. Only the biverticillate Penicillium spp. (P. funiculosum, P. islandicum, P. rubrum and P. tardum) produced substantial beta-galactofuranosidase after 1-4 weeks at 25 degrees C. No extracellular antigens of these four Penicillium spp. could be detected in culture filtrates by the sandwich ELISA technique when antibodies to the extracellular beta-galactofuranoside-containing polysaccharide antigen of P. digitatum was used. Antigens to all other Penicillium and Aspergillus spp. were easily detected in their culture filtrates.     

Improving the amylolytic activity of Saccharomyces cerevisiae glucoamylase by the addition of a starch binding domain

Glucoamylase produced by amylolytic strains of Saccharomyces cerevisiae (var. diastaticus) lacks a starch binding domain that is present in homologous glucoamylases from Aspergillus niger and other filamentous fungi. The absence of the binding domain makes the enzyme inefficient against raw starch and hence unsuitable for most biotechnological applications. We have constructed a hybrid glucoamylase-encoding gene by in-frame fusion of the S. cerevisiae STA1 gene and DNA fragment that encodes the starch binding domain of A. niger glucoamylase. The hybrid enzyme resulting from expression of the chimeric gene in S. cerevisiae has substrate binding capability and hydrolyses insoluble starch, properties not present in the original yeast enzyme.     

Analysis of glycosylinositol phosphorylceramides expressed by the opportunistic mycopathogen Aspergillus fumigatus

Acidic glycosphingolipid components were extracted from the opportunistic mycopathogen Aspergillus fumigatus and identified as inositol phosphorylceramide and glycosylinositol phosphorylceramides (GIPCs). Using nuclear magnetic resonance sppectroscopy, mass spectrometry, and other techniques, the structures of six major components were elucidated as Ins-P-Cer (Af-0), Manp(alpha1-->3)Manp(alpha1-->2)Ins-P-Cer (Af-2), Manp(alpha1-->2)Manp(alpha1-->3)Manp(alpha1-->2)Ins-P-Cer (Af-3a), Manp(alpha1-->3)[Galf(beta1-->6)]Manp(alpha1-->2)-Ins-P-Cer (Af-3b), Manp(alpha1-->2)-Manp(alpha1-->3)[Galf(beta1-->6)]Manp(alpha1-->2)Ins-P-Cer (Af-4), and Manp(alpha1-->3)Manp(alpha1-->6)GlcpN(alpha1-->2)Ins-P-Cer (Af-3c) (where Ins = myo-inositol and P = phosphodiester). A minor A. fumigatus GIPC was also identified as the N-acetylated version of Af-3c (Af-3c*), which suggests that formation of the GlcNalpha1-->2Ins linkage may proceed by a two-step process, similar to the GlcNalpha1-->6Ins linkage in glycosylphosphatidylinositol (GPI) anchors (transfer of GlcNAc, followed by enzymatic de-N-acetylation). The glycosylinositol of Af-3b, which bears a distinctive branching Galf(beta1-->6) residue, is identical to that of a GIPC isolated previously from the dimorphic mycopathogen Paracoccidioides brasiliensis (designated Pb-3), but components Af-3a and Af-4 have novel structures. Overlay immunostaining of A. fumigatus GIPCs separated on thin-layer chromatograms was used to assess their reactivity against sera from a patient with aspergillosis and against a murine monoclonal antibody (MEST-1) shown previously to react with the Galf(beta1-->6) residue in Pb-3. These results are discussed in relation to pathogenicity and potential approaches to the immunodiagnosis of A. fumigatus.     

Cloning and characterization of the phosphoglucomutase of Trypanosoma cruzi and functional complementation of a Saccharomyces cerevisiae PGM null mutant

Trypanosoma cruzi is the etiological agent of Chagas' disease, a chronic illness characterized by progressive cardiomyopathy and/or denervation of the digestive tract. The parasite surface is covered with glycoconjugates, such as mucin-type glycoproteins and glycoinositolphospholipids (GIPLs), whose glycans are rich in galactopyranose (Galp) and/or galactofuranose (Galf) residues. These molecules have been implicated in attachment of the parasite to and invasion of mammalian cells and in modulation of the host immune responses during infection. In T. cruzi, galactose (Gal) biosynthesis depends on the conversion of uridine diphosphate (UDP)-glucose (UDP-Glc) into UDP-Gal by an NAD-dependent reduction catalyzed by UDP-Gal 4-epimerase. Phosphoglucomutase (PGM) is a key enzyme in this metabolic pathway catalyzing the interconversion of Glc-6-phosphate (Glc-6-P) and Glc-1-P which is then converted into UDP-Glc. We here report the cloning of T. cruzi PGM, encoding T. cruzi PGM, and the heterologous expression of a functional enzyme in Saccharomyces cerevisiae. T. cruzi PGM is a single copy gene encoding a predicted protein sharing 61% amino acid identity with Leishmania major PGM and 43% with the yeast enzyme. The 59-trans-splicing site of PGM RNA was mapped to a region located at 18 base pairs upstream of the start codon. Expression of T. cruzi PGM in a S. cerevisiae null mutant-lacking genes encoding both isoforms of PGM (pgm1Delta/pgm2Delta) rescued the lethal phenotype induced upon cell growth on Gal as sole carbon source.     

Presence and regulation of the alpha-ketoglutarate dehydrogenase multienzyme complex in the filamentous fungus Aspergillus niger.

alpha-Ketoglutarate dehydrogenase has been demonstrated for the first time in cell extracts from the filamentous fungus Aspergillus niger. A minimum protein concentration of 5 mg/ml is necessary for detecting enzyme activity, but a maximum of ca. 0.060 mumol/min per mg of protein is observed only when the protein concentration is above 9 mg/ml. alpha-Ketoglutarate can partly stabilize the enzyme against dilution in the assay system. Neither bovine serum albumin nor a variety of substrates or effectors of the enzyme could stabilize the enzyme against inactivation by dilution. A kinetic analysis of the enzyme revealed Michaelis-Menten kinetics with respect to alpha-ketoglutarate, coenzyme A, and NAD. Thiamine PPi was required for maximal activity. NADH, oxaloacetate, succinate, and cis-aconitate were found to inhibit the enzyme; AMP was without effect. Monovalent cations including NH4+ were inhibitory at high concentrations (greater than 20 mM). The highest enzyme activity was found in rapidly growing mycelia (glucose-NH4+ or glucose-peptone medium). We discuss the possibility that citric acid accumulation is caused by oxaloacetate and NADH inhibition of the alpha-ketoglutarate dehydrogenase of A. niger.     

The effect of pH on glucoamylase production, glycosylation and chemostat evolution of Aspergillus niger

The effect of ambient pH on production and glycosylation of glucoamylase (GAM) and on the generation of a morphological mutant produced by Aspergillus niger strain B1 (a transformant containing an additional 20 copies of the homologous GAM glaA gene) was studied. We have shown that a change in the pH from 4 to 5.4 during continuous cultivation of the A. niger B1 strain instigates or accelerates the spontaneous generation of a morphological mutant (LB). This mutant strain produced approx. 50% less extracellular protein and GAM during both chemostat and batch cultivation compared to another strain with parental-type morphology (PS). The intracellular levels of GAM were also lower in the LB strain. In addition, cultivation of the original parent B1 strain in a batch-pulse bioreactor at pH 5.5 resulted in a 9-fold drop in GAM production and a 5-fold drop in extracellular protein compared to that obtained at pH 4. Glycosylation analysis of the glucoamylases purified from shake-flask cultivation showed that both principal forms of GAM secreted by the LB strain possessed enhanced galactosylation (2-fold), compared to those of the PS. Four diagnostic methods (immunostaining, mild methanolysis, mild acid hydrolysis and beta-galactofuranosidase digestion) provided evidence that the majority of this galactose was of the furanoic conformation. The GAMs produced during batch-pulse cultivation at pH 5.5 similarly showed an approx. 2-fold increase in galactofuranosylation compared to pH 4. Interestingly, in both cases the increased galactofuranosylation appears primarily restricted to the O-linked glycan component. Ambient pH therefore regulates both GAM production and influences its glycosylation.     

Comparative structural and molecular characterization of Streptococcus pneumoniae capsular polysaccharide serogroup 10

Streptococcus pneumoniae serogroup 10 includes four cross-reactive capsular polysaccharide (CPS) serotypes (10F, 10A, 10B, and 10C). In the present study, the structures of CPS10B and CPS10C were determined by chemical and high resolution NMR methods to define the features of each serotype. Both CPS10C and CPS10F had β1-6-linked Galf branches formed from the termini of linear repeating units by wzy-dependent polymerization through the 4-OH of subterminal GalNAc. The only difference between these polysaccharides was the wcrC-dependent α1-2 or wcrF-dependent α1-4 linkages between Gal and ribitol-5-phosphate. The presence of one linkage or the other also distinguished the repeating units of CPS10B and CPS10A. However, whereas these polysaccharides both had β1-3-linked Galf branches linked to GalNAc, only CPS10A had additional β1-6-linked Galp branches. These Galp branches and the reaction of a CPS10A-specific monoclonal antibody were eliminated by deletion of wcrG from the cps10A locus. In contrast, deletion of this gene from the cps10B locus had no effect on the structure of CPS10B, thereby identifying wcrG as a pseudogene in this serotype. The β1-3-linked Galf branches of CPS10A and CPS10B were eliminated by deletion of wcrD from each corresponding cps locus. Deletion of this gene also eliminated wcrG-dependent β1-6-linked Galp branches from CPS10A, thereby identifying WcrG as a branching enzyme that acts on the product of WcrD. These findings provide a complete view of the molecular, structural, and antigenic features of CPS serogroup 10, as well as insight into the possible emergence of new serotypes.     

Purification and properties of a 4-nitrophenylphosphatase from Aspergillus niger.

A 4-nitrophenylphosphatase (EC 3.1.3.41) was identified in extracts of Aspergillus niger. The production of this activity was decreased by growth on a phosphate-limiting medium and was greatest in a medium supplemented with corn steep liquor. The phosphatase activity was purified by hydrophobic, ion-exchange, and molecular sieve chromatography. The purified enzyme has a native size of approximately 80,000, polypeptide subunits with sizes of 37,000 upon denaturation, and a pI of 4.6. The activity was optimal at pH 8.0 and was stimulated by Mg2+ and to a lesser extent by Mn2+ but was inhibited by Zn2+ and Ca2+. The enzyme was highly specific for 4-nitrophenyl phosphate as substrate, having a Km of 0.77 mM and a turnover number of 108 s-1. The purified enzyme did not hydrolyze any of 22 sugar phosphates, mononucleotides, or other phosphocompounds tested. A small, but reproducible, amount of activity was measured using 5'-DNA phosphate as a substrate. Although some similarities exist to three previously characterized 4-nitrophenylphosphatases from Saccharomyces cerevisiae, the enzyme from A. niger is distinctly different from at least two of these activities.     

Hydrolytic activity of alpha-galactosidases against deoxy derivatives of p-nitrophenyl alpha-D-galactopyranoside

The four possible monodeoxy derivatives of p-nitrophenyl (PNP) alpha-D-galactopyranoside were synthesized, and hydrolytic activities of the alpha-galactosidase of green coffee bean, Mortierella vinacea and Aspergillus niger against them were elucidated. The 2- and 6-deoxy substrates were hydrolyzed by the enzymes from green coffee bean and M. vinacea, while they scarcely acted on the 3- and 4-deoxy compounds. On the other hand, A. niger alpha-galactosidase hydrolyzed only the 2-deoxy compound in these deoxy substrates, and the activity was very high. These results indicate that the presence of two hydroxyl groups (OH-3 and -4) is essential for the compounds to act as substrates for the enzymes of green coffee bean and M. vinacea, while the three hydroxyl groups (OH-3, -4, and -6) are necessary for the activity of the A. niger enzyme. The kinetic parameters (K(m) and Vmax) of the enzymes for the hydrolysis of PNP alpha-D-galactopyranoside and its deoxy derivatives were obtained from kinetic studies.     

Affinity chromatography of beta-glucosidase and endo-beta-glucanase from Aspergillus niger on concanavalin A-Sepharose: implications for cellulase component purification and immobilization

Affinity chromatography of a commercial preparation of beta-glucosidase from Aspergillus niger using concanavalin A-Sepharose (CAS) was employed as a means of purifying this glycoprotein. However, mannose (up to 1.08 M) was ineffective as an eluent of this enzyme from CAS, as were several other sugars and their derivatives, including 0.5 M glucose. Also, washing the CAS: beta-glucosidase complex with buffer at pH 3.5 in the absence of MnCl2 and CaCl2 (required to preserve the binding activity of concanavalin A below pH 5.0) did not result in elution of this enzyme. On the contrary, endoglucanase activity present in a crude cellulase complex (A. niger) which bound to CAS could be eluted by mannose (0.5-0.7 M) and was fractionated into at least two components. The CAS: beta-glucosidase complex hydrolyzed cellobiose to glucose and possessed an activity of 2,158 units/g dry CAS. It could be used, therefore, for continuous cellobiose hydrolysis without leakage of enzyme from the support.     

The ruminant parasite Haemonchus contortus expresses an α1,3-fucosyltransferase capable of synthesizing the Lewis x and sialyl Lewis x antigens

Glycoproteins from the ruminant helminthic parasite Haemonchus contortus react with Lotus tetragonolobus agglutinin and Wisteria floribunda agglutinin, which are plant lectins that recognize α1,3-fucosylated GlcNAc and terminal β-GalNAc residues, respectively. However, parasite glycoconjugates are not reactive with Ricinus communis agglutinin, which binds to terminal β-Gal, and the glycoconjugates lack the Lewis x (Lex) antigen or other related fucose-containing antigens, such as sialylated Lex, Lea, Leb Ley, or H-type 1. Direct assays of parasite extracts demonstrate the presence of an α1,3-fucosyltransferase (α1,3FT) and β1,4-N-acetylgalactosaminyltransferase (β1,4GalNAcT), but not β1,4-galactosyltransferase. The H. contortus α1,3FT can fucosylate GlcNAc residues in both lacto-N-neotetraose (LNnT) Galα1→4GlcNAcβ1→3Galβ1→4Glc to form lacto-N-fucopentaose III Galβ1→ 4[Fucα1→3]GlcNAcβ1→3Galβ1→4Glc, which contains the Lex antigen, and the acceptor lacdiNAc (LDN) GalNAcβ1→4GlcNAc to form GalNAcβ1→4[Fucα1 →3]GlcNAc. The α1,3FT activity towards LNnT is dependent on time, protein, and GDP-Fuc concentration with a Km 50 μ M and a Vmax of 10.8 nmol-mg?1 h?1. The enzyme is unusually resistant to inhibition by the sulfhydryl-modifying reagent N-ethylmaleimide. The α1,3FT acts best with type-2 glycan acceptors (Galβ1→4GlcNAcβ1-R) and can use both sialylated and non-sialylated acceptors. Thus, although in vitro the H. contortus α1,3FT can synthesize the Lex antigen, in vivo the enzyme may instead participate in synthesis of fucosylated LDN or related structures, as found in other helminths.     

Utilization of ram horn peptone in the production of glucose oxidase by a local isolate Aspergillus niger OC-3

Glucose oxidase (GO) is an enzyme that is used in many fields. In this study, ram horn peptone (RHP) was utilized as the nitrogen source and compared with other nitrogen sources in the production of GO by Aspergillus niger. To obtain higher GO activity, 14 A. niger strains were isolated from soil samples around Erzurum, Turkey. Among these strains, the isolate that was named A. niger OC-3 achieved the highest GO production. The production of GO was carried out in 100 mL scaled batch culture. The fermentation conditions such as initial pH, temperature, agitation speed, and time were investigated in order to improve GO production. The results showed that the cultivation conditions would significantly affect the formation of GO, and the utilization of the RHP achieved the highest enzyme production (48.6 U/mL) if compared to other nitrogen sources. On the other hand, the maximum biomass was obtained by using the fish peptone (7.2 g/L), while RHP yielded 6.4 g/L. These results suggest that RHP from waste ram horns could effectively be used in the production of GO by A. niger OC-3.